We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6×His, FLAG, 3×HA, 4×Myc, 10×Myc, GST, T7, TAP, were employed. Some carry the 2×35S-Ω promoter higher-level expression. The kanamycin- hygromycin-resistant markers are independently available each 43 types vectors, thus an additional transformation once-transformed plants can be carried out easily. Their small size high-copy...

Abstract Plants use nitrate, ammonium, and organic nitrogen in the soil as sources. Since elevated CO 2 environment predicted for near future will reduce nitrate utilization by C 3 species, ammonium is attracting great interest. However, abundant nutrition impairs growth, i.e., toxicity, primary cause of which remains to be determined. Here, we show that assimilation GLUTAMINE SYNTHETASE (GLN2) localized plastid rather than accumulation a challenges textbook knowledge. With exposure toxic...

Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance ions. We investigated the subcellular localization Arabidopsis thaliana AtMTP1, member CDF family, its physiological Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as 43 kDa protein vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level suspension-cultured cells not...

Abstract Geranylgeranyl diphosphate (GGPP) is the precursor for biosynthesis of gibberellins, carotenoids, chlorophylls, isoprenoid quinones, and geranylgeranylated proteins in plants. There a small gene family GGPP synthases encoding five isozymes one related protein Arabidopsis, all homologs have putative localization signal to translocate into specific subcellular compartments. Using synthetic green fluorescent (sGFP), we studied these synthases. When fusion were expressed by cauliflower...

The two guard cells of a stoma are produced by single symmetric division just before terminal differentiation. Recessive mutations in the FOUR LIPS (FLP) gene abnormally induce at least four contact with one another. These pattern defects result from persistence precursor cell identity that leads to extra divisions end lineage. FLP is likely be required for correct timing transition cycling encodes two-repeat (R2R3) MYB protein whose expression accumulates division. A paralogous gene, MYB88,...

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 TAP. Selection Arabidopsis transformants with BASTA was successfully carried out using both plate-grown soil-grown seedlings. Transformed rice calli suspension-cultured tobacco cells were...

Abstract Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity increased levels d-glucose. This suggests that coupling may be a novel sugar-signaling mechanism Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated vivo as Gα interaction partner functions downstream plasma membrane–delimited (GPA1) d-glucose signaling pathway. THF1 plastid protein localized both outer membrane and stroma. Contact...

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational modification than degradation. Here we describe identification of fission yeast, which show required a number nuclear events including control telomere length chromosome segregation. A disruption pmt3(+) gene, Schizosaccharomyces pombe SMT3, was not lethal, but mutant cells carrying disrupted gene grew slowly. The pmt3Delta showed...

Plants contain a large number of ATP-binding cassette (ABC) transporters belonging to different subclasses. AtPDR8 is the only member pleiotropic drug resistance (PDR) ABC transporter subclass in Arabidopsis that constitutively highly expressed. In transgenic plants harboring promoter fused β-glucuronidase (GUS), reporter expression was shown be strong stomata and hydathode. stomata, transcripts were particularly frequent cells surrounding air spaces. Subcellular fractionation immunochemical...

We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction chimeric genes between any promoter and cDNA. The structure R4pGWBs is almost the same as promoterless type improved pGWBs (ImpGWBs), except attR1 site replaced with attR4, which enables tripartite recombination these promoter- cDNA-entry clones. While ImpGWBs suitable analysis constitutive expression cDNAs in higher plants, have great advantage expressing...

Animal and yeast nucleolin function as global regulators of ribosome synthesis, their expression is tightly linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 the predominant if not only form protein found in most tissues, GFP-AtNuc-L1 fusion proteins were targeted nucleolus. Expression was strongly induced by sucrose or glucose but non-metabolizable mannitol 2-deoxyglucose. Sucrose also caused enhanced subunits C/D H/ACA small nucleolar...

Membrane trafficking to the plasma membrane (PM) is a highly organized process which enables plant cells build up their bodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) genes, encode proteins involved in trafficking, are much more abundant Arabidopsis genome than that of any other eukaryote. We have previously shown large number molecules cell localized predominantly on PM. In present study, order elucidate physiological function each PM-localized SNARE,...

The availability of complete plant genome sequences has opened a new era molecular research using approaches such as comprehensive expression analysis, ectopic and gene knockdown. For purposes, tools realizing easy rapid construction recombinant plasmids are required. Here, we describe Gateway cloning compatible binary vectors for post-genomic in genetic engineering. comprise variety reporters, epitope tags selective markers that should make them useful Agrobacterium-mediated transformation...

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of side chain isoprenoid quinones. We tried to construct an E. ispB-disrupted mutant but could not isolate chromosomal disrupted unless or its homolog was supplied on a plasmid. disruptants that harbored plasmids carrying homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 9, respectively. Our results indicate function essential normal...

Recent studies have revealed various functions for the small ubiquitin-related modifier (SUMO) in diverse biological phenomena, such as regulation of cell division, DNA repair and transcription, yeast animals. In contrast, only a limited number proteins been characterized plants, although plant SUMO are involved many physiological processes, stress responses, flowering time defense reactions to pathogen attack. Here, we reconstituted Arabidopsis thaliana SUMOylation cascade Escherichia coli....

The tonoplast K+ membrane transport system plays a crucial role in maintaining homeostasis plant cells. Here, we isolated cDNAs encoding two-pore channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco Two of the four variants NtTPK1 contained VHG GHG instead GYG signature sequence second pore region. All products were functional when expressed Escherichia coli cell membrane, was targeted to three promoter sequences N. active, expression these increased ∼2-fold by salt...

Abstract Cyclin D (CYCD) plays an important role in cell cycle progression and reentry response to external signals. Here, we demonstrate that Arabidopsis thaliana CYCD4 is associated with specific divisions the hypocotyl. We observed cycd4 T-DNA insertion mutants had a reduced number of nonprotruding cells stomata hypocotyl epidermis. Conversely, overexpression enhanced division files upper region hypocotyls, where are usually formed wild-type plants. The overproliferative were stomatal...

The pollen coat is a surface component of grains required for fertilization. To study how the produced, we identified and characterized recessive conditional male-sterile Arabidopsis mutant, flaky pollen1-1 (fkp1-1), whose lack functional coats. FKP1 single-copy gene in genome encodes 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMG-CoA synthase), an enzyme mevalonate (MVA) pathway involved biosynthesis isoprenoids such as sterols. We found that fkp1-1 possesses T-DNA insertion 550 bp...

Chimeric repressor gene-silencing technology (CRES-T) is a powerful tool that has recently been developed for the functional analysis of plant transcription factors and genetic manipulation traits. For CRES-T, factor converted to strong by fusion with an SRDX repression domain, which then expressed in plants induce loss-of-function phenotype. However, traditional CRES-T vectors are inconvenient gene cloning promoter exchange. In this study, we new efficient convenient use employing Gateway...

Schizosaccharomyces pombe produces ubiquinone-10 whose side chain is thought to be provided by the product generated decaprenyl diphosphate synthase. To understand mechanism of ubiquinone biosynthesis in S. pombe, we have cloned gene encoding synthase combination PCR amplification fragment and subsequent library screening. The determined DNA sequence gene, called dps, revealed that dps encodes a 378-amino-acid protein has typical conserved regions observed many polyprenyl synthases....

We investigated a nodulin 26-like protein NIP2;1, which belongs to the third subgroup of Arabidopsis aquaporins. Histochemical analysis promoter–β-glucuronidase fusion revealed root-specific expression NIP2;1 . The was detected in young roots, but not leaves, stems, flowers or siliques. transient linked with green fluorescent cultured cells showed its putative endoplasmic reticulum (ER) localization. expressed yeast had low water channel activity membranes. may function as and/or ER for...